Background
Identification of exosomal miRNA biomarkers in thyroid disease using a microfluidic platform
Thomas Haigh1,2, James England2, John Greenman1, Victoria Green1. 1 Daisy Research Laboratory, Castle Hill Hospital, University of Hull. 2 Department of Otolaryngology - Head and Neck Surgery, Hull University Teaching Hospitals NHS Trust
Introduction: Thyroid-derived exosomes contain microRNAs which have the potential to act as specific biomarkers in the diagnosis and monitoring of thyroid disease. Our team have an established method of maintaining live, human ex-vivo thyroid tissue within a “tissue‑on‑chip” device (Figure 1.). This chip enables the examination of exosomal microRNA biomarkers released directly from the ex-vivo thyroid tissue itself.
Aim: To determine differences in small extracellular vesicles (sEVs) released from thyroid cancer, Graves’ disease and benign thyroid tissue with a view to identifying biomarkers of disease.
Methods: Thyroid biopsies from twenty patients (Thyroid cancer n=11, Graves’ n=4, Hashimoto’s thyroiditis n=3, Benign n=2) have been divided into a minimum of 6 pieces and incubated on separate microfluidic devices for 6 days. Daily effluent has been collected and Nanoparticle tracking analysis (NTA) has provided EV size and concentration data. Small EVs have been isolated from the effluent by differential ultracentrifugation and Western blotting has demonstrated the expression of exosomal markers. RNA is currently being extracted from isolated exosomes ready for miRNASeq and qRT-PCR analysis.
Results: To date, NTA has detected nanoparticles throughout the full 6 days of tissue incubation in all samples of thyroid cancer, Graves’ and benign tissue microfluidic effluent (Figure 2.).
On the whole the mean size (nm) of the nanoparticles remains constant throughout the six day duration (130nm±22.8nm). Typically, the number of particles was greatest on day 1 and declined daily until around day 3. The tetraspanin exosomal marker CD63, was detected in both thyroid cancer and the Graves’ disease exosomal lysates investigated (Figure 3 and 4.).
CD81 was also detected in the Graves’ whole tissue specimen (Figure 4.) but not in the sEV lysate. Initial Nanodrop analysis has detected sufficient RNA in sEV isolated from a benign tissue, thyroid cancer and Graves’ disease microfluidic effluent which will now be processed for miRNASeq.
Conclusion: The microfluidic devices designed and manufactured in Hull, are able to maintain thyroid tissue for 6 days, during which time EVs are released and detected by NTA. The thyroid derived sEVs test positive for the exosomal markers, CD63 but not CD81. Further characterization of the sEV miRNA will help demonstrate whether or not thyroid pathologies possess a distinct profile which could be used as a biomarker of disease. This will have clinical relevance and application within the scope of thyroid disease diagnostics but also treatment monitoring and response.
- Celano M, et al., MicroRNAs as Biomarkers in Thyroid Carcinoma. Int J Genomics. 2017;6496570.
- Riley A, et al., A novel microfluidic device capable of maintaining functional thyroid carcinoma specimens ex vivo provides a new drug screening platform. BMC Cancer. 2019;19:259-271.
- Foster H. et al., Isolation and characterisation of Graves’ disease-specific extracellular vesicles from tissue maintained on a bespoke microfluidic device. Organs-on-a-chip 2021;3:100011.
Impact of ENT UK Foundation funding
As a group we are all extremely grateful for the ENT UK Foundation funding and support. This funding has enabled us to have the project up and running during its’ first year. The money has predominantly gone towards consumables required for the Western Blotting aspect of the project.
The funding has also contributed to Thomas Haigh being able to undertake a MD research project and learn lab-based techniques. Following Thomas Haigh being awarded a National Training Number the experience and skills obtained in the lab and academic environment will be invaluable for future participation in translational research projects.
Current Status
The research grant of £750 has been spent in entirety on consumables associated with the Western Blotting part of the project.
As detailed in the abstract, the project has in total run twenty samples of various thyroid pathology on the microfluidic platform to date. Nanoparticle tracking analysis has been performed on fourteen out of the twenty samples thus far along with Western Blotting which has demonstrated the presence of tetraspanin exosomal marker, CD63 within the microfluidic effluent of the thyroid tissue. The funding provided by ENT UK has confirmed the presence of exosomes in the thyroid effluent released from tissue incubated on the microfluidic device. As a direct result, further work is on-going to extract RNA from the isolated exosomes released from the thyroid tissue and sequence the miRNAs present. This will elucidate whether different thyroid pathologies have varying EV characteristics with unique molecular profiles which have the potential to be used as biomarkers of disease. This will have clinical relevance and application within the scope of thyroid disease diagnostics but also in both treatment monitoring and response.
The project has been fortunate to receive further funding from the British Association of Endocrine and Thyroid Surgeons (BAETS) Research Grant for £5,000 to help carry on the sequencing work and received invitation to present at the free paper sessions at the upcoming annual meeting in October. The work is also due to be presented as a podium presentation at the North of England ENT Society - Autumn Meeting taking place in Leeds.
The attached abstract has also been submitted for consideration by the University of Hull selection committee to be presented at the 19th International Medical PhD. Conference 2022 – ‘From bench to bedside’ at the Faculty of Medicine in Hradec Kralove, Czech Republic.
The aim for completion of the entire project is August 2023. This will be accompanied by a MD thesis write up by Thomas Haigh and hopefully a publication in a reputable scientific journal. Foundations have also been put in place for further research by the taking of blood plasma samples at the time of the thyroid surgeries. Relevant ethical approval has been granted for this. The next step therefore, with further funding, would be to correlate the findings in the different thyroid pathologies microfluidic effluent microRNA profile with that of the respective patient’s plasma samples.
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